This invention relates generally to the field of recombinant retroviral particles for use in gene delivery, e.g., for use in gene therapy.
Retroviruses are enveloped RNA viruses that, after infection of a host cell, reverse transcribe their RNA genomes into a DNA intermediate, or provirus. The provirus can be stably integrated into the host""s cellular DNA. Gene products encoded by the provirus are then expressed by the host cell to produce retroviral virions, thereby replicating the virus. Because the retroviral genome can be manipulated to include exogenous nucleotide sequence(s) of interest for expression in a target cell, retroviral vectors are important tools for stable gene transfer into mammalian cells. Many proposed gene therapy applications use retroviral vectors to take advantage of the ability of these naturally infectious agents to transfer and efficiently express recombinant nucleotide sequences in susceptible target cells (see, e.g., Miller 1992 Nature 357:455-460; Miller Curr. Top. Microbiol. Immunol. 158:1-24). Retroviral vectors suitable for use in such applications are generally defective retroviral vectors that are capable of infecting the target cell, reverse transcribing their RNA genomes, and integrating the reverse transcribed DNA into the target cell genome, but are incapable of replicating within the target cell to produce infectious retroviral particles (e.g., the retroviral genome transferred into the target cell is defective in gag, the gene encoding virion structural proteins, and/or in pol, the gene encoding reverse transcriptase).
Use of retroviral vectors is limited in many aspects. For example, although retroviruses can efficiently infect and stably integrate into the genome of rapidly-dividing cells, retroviral integration into the genome of non-dividing or slowly dividing cells is inefficient (Springett et al. 1989 J. Virol. 63:3865-3869; Miller et al. 1990 Mol. Cell. Biol. 10:4239-4242; Roe et al. 1993 EMBO J. 12:2099-2108). Most packaging systems provide only modest vector titers, and the fragility of retroviral vector particles complicate purification and concentration (Paul et al. 1993 Hum. Gene Therap. 4:609-615). Finally, retroviruses enter target cells by binding of retroviral envelope glycoproteins (encoded by the env gene) to specific target cell surface receptors. This envelope protein-cell surface receptor interaction is often species specific, and in some cases even tissue specific. Moreover, the level of expression of the cell surface receptor on the target cells can vary widely among target cells. As a result, retroviruses usually have a limited host range (Kavanaugh et al. 1994 Proc. Natl. Acad. Sci. USA 91:7071-7075; Hopkins 1993 Proc. Natl. Acad. Sci. USA 90:8759-8760).
One strategy for both expanding retroviral host cell range and increasing the structural stability of the retroviral virion involves production of pseudotyped retroviral viral vectors. Pseudotyped retroviral vectors useful in transformation of target cells are generally composed of retroviral virion structural proteins (e.g, Gag proteins), a recombinant RNA genome containing the nucleotide sequence of interest, the Pol protein for reverse transcription of the recombinant RNA contained in the virion, and a non-retroviral envelope protein or an envelope protein from a different retrovirus. The recombinant RNA genome is usually replication defective, e.g., defective in the pol and/or gag genes, to prevent production of infectious retrovirus following transfer of the nucleotide sequence of interest into the target cell. The envelope protein of the pseudotyped retrovirus is normally selected to provide a broader host range or to provide selective targeting of cells to be infected.
The envelope protein of vesicular stomatitis virus (VSV), termed VSV G, is a strong candidate for use in the production of pseudotyped retroviral vectors. VSV G can infect a variety of cell types from a wide range of mammalian and non-mammalian species, including humans, hamsters, insects, fish, and frogs, with a greater efficiency than traditional amphotropic retroviral vectors. The putative receptor(s) for VSV include phosphatidyl serine, phosphatidyl inositol and/or GM3 ganglioside (Mastromarino, et al., 1987 J. Gen. Virol. 68:2359-2369; Conti, et al., 1988 Arch. Virol. 99:261-269), all of which are ubiquitous and abundant components of plasma membrane. VSV G pseudotyped retroviral vectors have enhanced structural stability allowing for concentration to titers of greater than 109/ml by ultracentrifugation. (Emi et al. 1991 J. Virol. 65:1202-1207; Yee et al., 1994 Proc. Natl. Acad. Sci. USA 91:9564-9568; Burns et al. 1993 Proc. Natl. Acad. Sci. USA 90:8033-8037,; Lin et al. 1994 Science 265:666-669). When expressed in packaging cells, VSV G efficiently forms pseudotyped virions with the genome and core components derived from retroviruses such as murine leukemia virus (MuLV). Packaging cell lines that express the retroviral gag and pol genes and-the VSV G envelope protein produce pseudotyped retroviral particles having the retroviral Gag and Pol proteins enclosed in a VSV G-containing envelope (see FIG. 1), resulting in the production of virions whose infectivity is blocked by anti-VSV G antibodies (Emi et al. 1991 supra; Yee et al. 1994 supra). These properties of VSV G pseudotyped virions not only expand the use of retroviral vectors for genetic studies in previously inaccessible species, but also facilitate more efficient pre-clinical and clinical studies of the potential for human gene therapy.
However, production of VSV G pseudotyped retroviral virions has met with several difficulties. First, VSV G is cytotoxic. High level expression of VSV G in mammalian cells leads to syncytia formation and cell death, making it difficult to establish stable cell lines expressing VSV G (Yee et al. 1994 supra; Burns et al. 1993 supra). Pseudotyped VSV G virions have been produced by transient expression of the VSV G gene after DNA transfection of 293GP cells expressing the Gag and Pol components of MULV, yielding vector preparations having titers of 105-106/ml (Yee et al 1994 supra). However, generation of VSV G pseudotyped virions by transient VSV G expression is cumbersome, labor intensive, and unlikely to be amenable to clinical applications that demand reproducible, certified vector preparations.
Several inducible promoter systems have been described including those controlled by heavy metals (Mayo et al. 1982 Cell 29:99-108), RU-486 (a progesterone antagonist) (Wang et al. 1994 Prxoc. Natl. Acad. Sci. USA 91:8180-8184), steroids (Mader and White, 1993 Proc. Natl. Acad. Sci. USA 90:5603-5607), and tetracycline (Gossen and Bujard 1992 Proc. Natl. Acad. Sci. USA 89:5547-5551; U.S. Pat. No. 5,464,758). However, heavy metals are toxic to cells, compromising the use of this inducible promoter system. The inducible promoter of the RU-486 system is significantly expressed in the absence of RU-486 and is induced only 10- to 20-fold in the presence of RU-486 (Wang et al. 1994), making this system undesirable for expression of VSV G for production of pseudotyped retroviral virions.
The tetracycline-inducible system of Gossen and Bujard has been used to regulate inducible expression of several genes (Gossen and Bujard 1992, supra; Furth et al. 1994 Proc. Natl. Acad. Sci. USA 91:9302-9306; Howe et al. 1995 J. Biol. Chem. 270:14168-14174; Resnitzky et al. 1994 Mol. Cell. Biol. 14:1669-1679; Shockett et al. 1995 Proc. Natl. Acad. Sci. USA 92:6522-6526). This system uses a chimeric transcription factor, termed tTA, which is composed of the repressor of Escherichia coli (E. coli) tetracycline-resistance operon (tetR) and the activation domain (carboxyl terminal domain) of virion protein 16 (VP16) of herpes simplex virus (HSV) (Triezebberg et al. 1988 Genes Dev. 2:718-729). The gene of interest is placed downstream of a minimal cytomegalovirus (CMV) 1A promoter, derived from the immediate early CMV genes, which is linked to multiple copies of tetO, the binding site for the tetracycline repressor tetR. In the absence of tetracycline, the tetR portion of the transactivator binds the tetO sequences of the promoter and the VP16 portion facilitates transcription. When tetracycline is present, tetracycline binds the tetR portion of tTA, which in turn prevents binding of the tetR portion to the tetO sequence(s) of the promoter, thus inhibiting transcription. Since even low concentrations of tetracycline are sufficient to block tTA function, and since most mammalian cells can tolerate tetracycline, this system provides a tightly regulated on/off switch for gene expression that can be controlled by varying the tetracycline concentration to which the cells are exposed. However, establishment of cell lines stably expressing large amounts of the tetracycline-transactivator (tTA) is difficult, since the VP16 activation domain decreases, or xe2x80x9csquelches,xe2x80x9d general cellular transcription when expressed in large quantities in mammalian cells (Gossen and Bujard 1992 supra Gossen and Bujard 1992, supra; Shockett et al. 1995 supra; Gill et al. 1988 Nature 334:721-724; Ptashne et al. 1990 Nature 346:329-331). Thus, the tTA inducible expression system is not desirable for production of VSV G pseudotyped retroviral vectors.
There is a clear need in the field for an inducible expression system useful in the production of cytotoxic gene products, such as VSV G, and useful in the production VSV G pseudotyped retroviral vectors.
The present invention features compositions and methods for the inducible expression of a polypeptide, especially a polypeptide normally cytotoxic to the eukaryotic host cell in which it is to be expressed. A nucleotide sequence encoding a polypeptide of interest is operably linked to an inducible promoter (e.g, a promoter composed of a minimal promoter linked to multiple copies of tetO, the binding site for the tetracycline repressor (tetR) of the Escherichia coli tetracycline resistance operon Tn10). Expression from the inducible promoter is regulated by a multi-chimeric transactivating factor, composed of a first ligand-binding domain that negatively regulates transcription (e.g., a prokaryotic tetracycline repressor polypeptide), a transcriptional activation domain, and a second ligand-binding domain that positively regulates the transcriptional activation function of the transactivator (e.g., a ligand-binding domain of a steroid receptor, preferably an estrogen receptor (ER)). Transcription of the nucleotide sequence under control of the inducible promoter is activated by the multi-chimeric transactivator when both the ligand that binds the first ligand-binding domain (e.g., tetracycline) is absent and the ligand that binds the second ligand-binding domain (e.g., a steroid) is present. This inducible expression system is particularly useful in the expression of the cytotoxic protein VSV G for the production of pseudotyped retroviral vectors.
A primary object of the invention is to provide a system for inducible expression of a polypeptide in a eukaryotic cell, particularly a polypeptide that is cytotoxic to the host cell when expressed at normal to high levels.
Another object of the invention is to provide a system for inducible expression of a polypeptide that utilizes a transactivating factor that does not significantly affect general cellular transcription.
Yet another object of the invention is to provide an inducible expression system that can be used to generate packaging cell lines that provide high titers of VSV G pseudotyped retroviral vectors.
Another object of the invention is to provide VSV G pseudotyped retroviral vectors having an expanded host range to allow transformation of species, including non-mammalian species previously inaccessible using conventional transformation techniques.
An advantage of the present invention is that any nucleotide sequence can be expressed using the inducible expression system of the invention.
Another advantage is that the transcriptional activation function of the transactivating factor is substantially inactive until exposed to steroid (or an analog thereof), thus decreasing any possible interference with general cellular transcription.
Another advantage is that activation of the inducible expression system requires two independent signals (i.e., the absence of tetracycline and the presence of estrogen), thus reducing the incidence of undesired transcriptional activation (e.g., transcriptional activation of the sequence operably linked to the inducible promoter under conditions other than the absence of tetracycline and the presence of steroid).
These and other objects, advantages and features of the present invention will become apparent to those persons skilled in the art upon reading the details of the vectors, cell lines and methodology as more fully set forth below.